The broad, long-term objective of this proposal is to define the molecular mechanisms of action and regulation of the par stability determinant encoded on the Enterococcus faecalis plasmid pAD1. par is no larger than 404 nucleotides and encodes 2 small RNAs essential for its function. Evidence indicates that par functions as a post-segregational killing system. The larger of the 2 RNAs, RNA1, is the toxic component of the system and encodes and 33 amino acid peptide. The smaller RNA, RNA, RNAII, regulates RNAI activity. It is hypothesized that RNAII inhibits peptide translation as an antisense RNA and that the RNAI- encoded peptide inhibits some essential host function.
Four specific aims will be pursued to test this hypothesis. First, detailed structural studies on both par RNAs and their interaction complex will be performed in vitro and in vivo. Second, potential autoregulatory effects on par RNA production will be examined by a variety of genetic approaches. Third, the toxin target will be determined by identifying host proteins that interact with the RNAI-encoded peptide. Fourth, other plasmid- encoded stability determinants that interact with par will be identified by conducting competition experiments with various constructs and by characterizing a potential active partition system located adjacent to par. It is hoped that the par system could be manipulated in some way to provide a useful antibacterial treatment for enterococci infection. Alternatively, identification of the par toxin target could provide a useful target for the design of new antibiotics effective against the enterococci.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM055544-01A2
Application #
2848499
Study Section
Bacteriology and Mycology Subcommittee 2 (BM)
Project Start
1999-05-01
Project End
2002-04-30
Budget Start
1999-05-01
Budget End
2000-04-30
Support Year
1
Fiscal Year
1999
Total Cost
Indirect Cost
Name
University of South Dakota
Department
Other Basic Sciences
Type
Schools of Medicine
DUNS #
929930808
City
Vermillion
State
SD
Country
United States
Zip Code
57069
Weaver, Keith E (2015) The Type I toxin-antitoxin par locus from Enterococcus faecalis plasmid pAD1: RNA regulation by both cis- and trans-acting elements. Plasmid 78:65-70
Brinkman, Cassandra L; Bumgarner, Roger; Kittichotirat, Weerayuth et al. (2013) Characterization of the effects of an rpoC mutation that confers resistance to the Fst peptide toxin-antitoxin system toxin. J Bacteriol 195:156-66
Weaver, Keith E (2012) The par toxin-antitoxin system from Enterococcus faecalis plasmid pAD1 and its chromosomal homologs. RNA Biol 9:1498-503
Johnson, Christopher M; Manias, Dawn A; Haemig, Heather A H et al. (2010) Direct evidence for control of the pheromone-inducible prgQ operon of Enterococcus faecalis plasmid pCF10 by a countertranscript-driven attenuation mechanism. J Bacteriol 192:1634-42
Shokeen, Sonia; Johnson, Christopher M; Greenfield, Tony J et al. (2010) Structural analysis of the Anti-Q-Qs interaction: RNA-mediated regulation of E. faecalis plasmid pCF10 conjugation. Plasmid 64:26-35
Weaver, Keith E; Kwong, Stephen M; Firth, Neville et al. (2009) The RepA_N replicons of Gram-positive bacteria: a family of broadly distributed but narrow host range plasmids. Plasmid 61:94-109
Shokeen, Sonia; Greenfield, Tony J; Ehli, Erik A et al. (2009) An intramolecular upstream helix ensures the stability of a toxin-encoding RNA in Enterococcus faecalis. J Bacteriol 191:1528-36
Weaver, Keith E; Reddy, Shirisha G; Brinkman, Cassandra L et al. (2009) Identification and characterization of a family of toxin-antitoxin systems related to the Enterococcus faecalis plasmid pAD1 par addiction module. Microbiology 155:2930-40
Shokeen, Sonia; Patel, Smita; Greenfield, Tony J et al. (2008) Translational regulation by an intramolecular stem-loop is required for intermolecular RNA regulation of the par addiction module. J Bacteriol 190:6076-83
Francia, Maria Victoria; Weaver, Keith E; Goicoechea, Patricia et al. (2007) Characterization of an active partition system for the Enterococcus faecalis pheromone-responding plasmid pAD1. J Bacteriol 189:8546-55

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